PROJECT ABSTRACT Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people?s risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which are the first line of defense against infectious pathogens in the lungs. We have used primary airway epithelial cells isolated from healthy and alcoholic patients and differentiated them in vitro to examine the impact of alcohol on cell barrier function and morphology. Healthy differentiated primary airway epithelial cells exhibit a cobblestone- like pattern and become immobile once the monolayer has matured? a normal airway cell phenotype known as ?jamming?. By contrast, airway epithelial cells isolated from chronic alcoholics remained migratory and in an ?unjammed? state, since areas of stretched cells and swirls of cells appear in the monolayer. Additionally, rat tracheal cells grown and differentiated in vitro in the presence of ethanol remain unjammed while control monolayers become jammed. It is known that the transmembrane protein junctional adhesion molecule A (JAM- A) regulates epithelial cell migration, and it also regulates barrier function by controlling the paracellular flow of small molecules as part of tight junction complexes. Recently, we found that both rat and human in vitro differentiation models chronically exposed to alcohol have decreased JAM-A expression at both RNA and protein levels and decreased barrier function compared to their control counterparts. Nevertheless, how chronic alcohol exposure decreases JAM-A expression and impacts airway epithelial barrier function and migration has not been fully elucidated. Chronic alcohol exposure is known to activate TGF-?1 and recent evidence has linked TGF-?1 to repression of JAM-A expression. Thus, we hypothesize that the deleterious effects of chronic alcohol exposure on conducting airway epithelial cells are due to TGF-?1 activation causing attenuation of JAM- A expression and subsequent unjamming. My research plan is designed to determine how chronic alcohol exposure (potentially through TGF-?1) impacts JAM-A expression and, therefore, barrier function in primary airway epithelial cells (Aim 1), and to define how JAM-A regulates collective cell migration/cell jamming (Aim 2). The goal of this project is to identify targetable pathways by which chronic alcohol exposure causes barrier function and cell migration defects in conducting airway epithelial cells.